Plant and Soil 228: 253264, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands.

253

Low-P tolerance by maize (Zea mays L.) genotypes: Signicance of root growth, and organic acids and acid phosphatase root exudation
Alain Gaume1 , Felix Mchler1 , Carlos De Le on2 , Luis Narro2 & Emmanuel Frossard1,3
of Plant Sciences, Swiss Federal Institute of Technology (ETH Zurich), Eschikon 33, Postfach 185, CH8315 Lindau, Switzerland. 2 CIMMYT regional ofce at CIAT, A. A. 6713, Cali, Colombia. 3 Corresponding author
Received 15 February 2000. Acceped in revised form 21 August 2000
1 Institute

Key words: acid phosphatase, low-P tolerance, organic acids, root exudation, Zea mays L. genotypes

Abstract We investigated some mechanisms, which allow maize genotypes to adapt to soils which are low in available P. Dry matter production, root/shoot-ratio, root length and root exudation of organic acids and acid phosphatase were investigated in four maize genotypes grown under P-decient and P-sufcient conditions in sterile hydroponic culture. A low-P tolerant, an acid-tolerant and a low-P susceptible genotype of maize were compared with a Swiss commercial cultivar. The study found increased root development and increased exudation of acid phosphatase under P-decient conditions in all maize genotypes, except for the Swiss cultivar. Effects on root formation and acid phosphatase were greater for the low-P tolerant than for the low-P susceptible, and the acid soil tolerant genotypes. Organic acid contents in root tissues were increased under P deciency and related to increased PEPC activity. However, the increase in contents was associated with an increase in exudation for the low-P tolerant genotype only. The low-P susceptible genotype was characterized by high organic acid content in roots and low organic acid exudation. The organic acids content in the phloem exudates of shoots was related to root exudation under different P supply, to the difference between lines in organic acids root content, but not to the low-P tolerance or susceptibility of maize genotypes.

Introduction Low-phosphorus (P) availability strongly limits plant productivity in tropical soils. Plant adaptations allowing for an improved growth in low P soils are related to the ability of a plant to take up more P from a decient soil (higher P acquisition efciency), or/and to its ability to produce more dry matter for a given quantity of P (higher P use efciency) (Marschner, 1995; Raghothama, 1999). A higher P acquisition efciency can be related (a) to the development of a more extensive root system, in association or not with mycorrhizal fungi, or specic specialized roots such as proteoid roots or root hairs (McCully, 1999; Raghothama, 1999), allowing the plant to explore a larger volume of soil, and (b) to changes in root physiology
FAX No: 523549119.

E-mail: emmanuel.frossard@ipw.agrl.ethz.ch

allowing the uptake of P at lower concentrations in the soil solution, and/or the uptake of P from insoluble inorganic or organic forms (Marschner, 1995). Modication of root growth and architecture is a well-documented response to P starvation (Lynch, 1995; Mollier and Pellerin, 1999). Authors generally agree that P deciency in maize leads to a higher root/shoot ratio (Anghinoni and Barber, 1980; Rosolem et al., 1994). Effects of P deciency on root biomass and root length are more controversial. Anghinoni and Barber (1980), using a P starvation experiment, found increased root length and dry weight in 12 day-old maize plants. By contrast, Khamis et al. (1990) observed no effect of P deprivation on maize root biomass. When phosphate is taken up by plants, it is transferred from the solid phase of the soil to the roots via the rhizosphere (soil/root interface), a zone where up to 30% of the plant photosynthetates may be exuded

254 by the roots (Lynch and Whipps, 1989). Excretion of organic acids, enzymes and protons by roots may play a major role in the P nutrition of various crops (Raghothama, 1999; Uren and Reisenauer, 1988). The competition of phosphate and organic anions for similar adsorption sites can increase the concentration of P in the soil solution (Gerke, 1992; Staunton and Leprince, 1996). Nagarajah et al. (1970) and Jones and Darrah (1994b) showed that the efciency with which organic acids desorb P from iron-oxide and clay minerals, or prevent the sorption of newly-added P, decreased in the order tri-, di- and monocarboxylic oganic acids. Root exudation of malic and succinic acids were increased in radish and rape plants under P starvation (Zhang et al., 1997). Increased exudation of citric acid by white lupin under P-decient conditions (Johnson et al., 1996) led to a higher release of inorganic phosphate from phosphated ferric hydroxide (Gardner et al., 1983) and from a pool of soil phosphorus unavailable to soybean (Braum and Helmke, 1995). Gerke (1992) showed that citric acid also liberates P complexed by soil organic matter. In white lupin, Johnson et al. (1996) demonstrated that increased citric and malic acid secretion from proteoid roots under P deciency was correlated with the increased activity of several enzymes involved in organic acid synthesis, including phosphoenolpyruvate carboxylase (PEPC). While the secretion of carbohydrates and amino acids by maize roots increased under P-decient conditions (Jones and Darrah, 1994a; Matsumoto et al., 1979), little is known about the inuence of P nutrition on the release of organic acids from maize roots. Tarafdar and Jungk (1987) measured acid phosphatase activity in the rhizosphere of wheat and clover corresponding to a depletion of soil organic phosphates across this zone. Their work suggests that rhizosphere phosphatases play an important role in the release of Pi from organic soil P, for subsequent uptake by plants. Enhancement of acid phosphatase activity with phosphate starvation has been demonstrated for maize (Helal and Sauerbeck, 1987; Kummerova, 1986). This research was conducted to elucidate some of the putative mechanisms governing P acquisition efciency in maize. To achieve this, four lines (a lowP tolerant line from Thailand, acid soil-tolerant and low-P susceptible lines from Colombia, and a Swiss cultivar bred in P-rich soils) were grown in hydroponic culture with the roots maintained under sterile conditions, and the effect of P starvation on plant growth, root growth and the exudation of organic acids and acid phosphatase by roots was studied.

Materials and methods Plant material Four genotypes of maize (Zea mays L.) were selected for the study. These were as follows: NST90201 (S) CO-422-2-3-1-7. An inbred line derived from a triple hybrid developed by the Thai Department of Agriculture. This inbred has been selected as tolerant to low-P conditions. SA3-C4HC (1625)-2-4-9-7-B-B-B-B-1. An inbred line developed by CIMMYT-Colombia from the acid soil tolerant population SA3. This inbred is susceptible to low-P conditions. ICA V-110 Sikuani. An open pollinated variety developed in Colombia by recombining selected acid soil-tolerant lines derived from Population SA3 (Friesen et al., 1997). Corso. A swiss genotype selected in high P soils for fast development and high dry matter production in the rst vegetative growth stages. NST, SA3 and Sikuani were provided by the CIMMYT regional ofce at CIAT, Cali, Colombia. Corso was provided by UFA Seed Company, Switzerland. Growth conditions Seeds of 0.280.30 g weight were surface sterilized (30 s in 18 m H2 SO4 , 5 min in 95% ethanol, and 30 min in 10% H2 O2 , washing with sterile water after each treatment), germinated on agar plates (4 d in the dark at room temperature) and transferred to culture vessels under sterile conditions. A culture vessel consisted of a Pyrex tube (50 cm long, 5 cm diameter) with a rubber cap on the bottom and an aluminum cap on the top. One germinated seed was placed on a net of Teon (0.4 cm diameter mesh), xed 6 cm below the top end of the tube. Nutrient solution was contained in the tube up to 2.0 cm below the Teon-net. Nutrient solution and culture vessel had been autoclaved separately before the seed was added. Nutrient solutions were modied from Hoagland and Arnon (1938) and consisted of mgSO4 (1 mM); Ca(NO3 )2 (2.5 mM); Fe-EDTA (0.1 mM); H3 BO3 (0.01 mM); MnSO4 (0.001 mM); ZnSO4 (0.001 mM); CuSO4 (0.0005 mM); Na2 MoO4 (0.0005 mM). The + P treatment contained normal nutrient solution with 0.75 mM K2 SO4 and 1 mM KH2 PO4 , while the P treatment

255 was amended with 1.25 mM K2 SO4 only. The pH of the nutrient solution was adjusted to 5.5 and was not affected by plant growth as showed by Bertrand (1998). After 5 days of plant growth, when seedlings had been established, aluminum caps were removed and the Teon-net was overlayered with 0.51.0 cm of sand (0.50.7 cm particle size) and a mixture of liquid parafn (solid below 5153C, Merck) and Vaseline (Maino Pharm AG) leaving the roots in the sterile nutrient solution and the shoots exposed to the open air. Air was introduced into the nutrient solution at the bottom through a sterile lter (0.2 m, Millipore) and released at the top through a silicone hose crossing the parafn layer and a second sterile lter. Nutrient solution was changed every 3 d through the bottom of the tube. Each manipulation with the culture vessels was conducted using aseptic technique. Plants were grown in a controlled environment with a photosynthetic photon ux density of 250 mol quanta m2 s1 during a 16 h photoperiod and a day/night-temperature of 23/18 C. Determination of growth characteristics Dry matter, P content in root and shoot and length of the 3 longest roots of each plant were determined in 18-day old seedlings. Dried plant parts were homogenized and samples (250 mg) were ignited at 540 C in an oven for 5 h. Residues were extracted in 6.5 N HCl and analyzed for P according to John (1970). Preparation of samples for analysis of organic acid contents and PEP Carboxylase activity Root extracts Root tips (segment A; 01.5 cm), and segments at 5 cm distance from root tips (segment B; 56.5 cm) were sampled. Twenty root fragments (50150 mg fresh weight) for each root part were weighted and kept on ice, and ground with a pestle in a cold mortar, with 1.5 ml CO2 -free buffer (100 mM Tris, 10 mM mgCl2 , pH 8.0) and 0.5 g sea sand. After centrifugation (14 000 g, 2 min) organic acid content and PEPC activity were determined in the supernatant. Exudation samples Phloem sap was collected according to the modied method of King and Zeevaart (1974). Shoots were cut 0.5 cm above the basis and dipped in 1 mM Na-EDTA to discard xylem exudates. After 10 min, phloem sap was collected in 50 ml 1 mM Na-EDTA for 2 h. To measure organic acids exuded by whole roots, samples of nutrient solution were collected 3 h after nutrient solution in the culture vessels were renewed. The exudation by specic root parts was examined by transferring plants into a 5 cm high PVC box (25 cm 15 cm 5 cm) lled with nutrient solution. Specic parts of the intact root (segment A and B) were dipped for 3 h into a plastic cap (1.5 cm high and 4 cm ), lled with the specic nutrient solution and containing 0.25 mg benzylpenicillin potassium salt ml1 (Fluka ref. 13750) to prevent bacterial growth. The exudation samples were tested for sterility on agar plates at room temperature and 37 C, and stored at 20 C until required for analysis of organic acids. Organic acid analyses Organic acids were analyzed by ion chromatography using a Dionex DX500 system. An anion exclusion column Ion Pac ICE-AS6 was used in combination with an anion-ICE micromembrane suppressor. The eluent was 1 mM uorobutyric acid and had a ow rate of 1 ml/min. The regenerant for the suppressor was 5 mM tetrabutylammonium hydroxide and had a ow rate of 4 ml/min. Suppressed conductivity was detected. Samples were acidied (100 l 1 N HCl added to 10 ml sample) and purged with nitrogen (N2 ) in order to lower carbonate concentration. Undiluted samples (50 l) were injected and analyzed. This method was not appropriate for oxalic acid, which coeluted with inorganic anions, or for long-chain carbonic acids (butyric acid etc.), which were not eluted in this system. Samples were tested for oxalic acid using an anionexchange column Ion Pac AS10 in combination with a suppressor ASRS II with 50 mM NaOH as eluent. Oxalic acid contents were very low and are not reported. Determination of PEP carboxylase (PEPC) activity in roots The PEPC assay was conducted in a spectrophotometer at 25 C according to the NADH-linked method (Vance et al., 1983). The nal volume of the reaction mixture was 1 ml and contained 25100 l root extract in CO2 -free extraction buffer, 2 mM phosphoenolpyruvate (PEP), 0.14 mM nicotinamide adenine dinucleotide (NADH), and 5 Units malate dehydrogenase (MDH). The reaction was initiated by adding 5 mM HCO3 and the decrease in absorption at 340 nm was recorded.

256 Determination of acid phosphatase activity Activity released into solution Nutrient solution in the culture vessels was replaced by sterile distilled water containing 1 mM CaCl2 . The activity of acid phosphatase was assayed after 24 h of root exudation, using p-nitrophenyl phosphate (pNPP) as a substrate (Tabatabai, 1982). An aliquot of solution adjusted to a total volume of 5 ml with distilled water was added to 0.5 ml of the Modied Universal Buffer (pH 5.0) and 1 ml of 0.025 m pNPP in reagent tubes. Tubes were maintained at 37 C for 1 h and the reaction was terminated by the addition of 20 ml of 0.5 M NaOH. The absorbance at 410 nm was measured to determine the amount of released p-nitrophenol (pNP). Phosphatase activity was expressed in terms of Units (U). One Unit of acid phosphatase is the amount of enzyme which hydrolyses 1.0 mol of p-nitrophenyl phosphate per min at 37 C. Activity adhering to the roots Roots were incubated for 30 min at 34 C in sterile distilled water containing 100 mM NaCl, in order to collect the acid phosphatase adhering to the epidermal cell layers of the roots. The activity of acid phosphatase released into the solution was determined as described above. Statistical background Statistical analyses of data were carried out by ANOVA tests. Signicance was assigned at p < 0.05 with Duncans test. All the analyses were performed using the SYSTAT statistical package (SYSTAT, 1994). nutrient uptake in this low-P tolerant genotype. Relatively small changes with P deciency were found for the low-P susceptible SA3 and for Corso. The acid soil-tolerant Sikuani responded similarly to NST to P starvation (Table 1). P deciency increased the anthocyanin red coloration in the maize genotypes, whereas in the presence of P, leaves of the four genotypes remained green (Table 2). Coloration was especially strong in the low-P tolerant NST and the acid soil-tolerant Sikuani. Anthocyanin formation may be a protective mechanism against oxygen free radical stress induced in P-decient illuminated leaves (Hrazdina and Zobel, 1991; Schopfer, 1984). It is suggested that anthocyanin formation may contribute to low-P tolerance of maize genotypes. P content in seedlings P contents in 18-d old maize seedlings grown in Pdecient nutrient solution were lower than the amount of P measured in seeds (1.14 0.08 mg P/seed across the four genotypes), suggesting that part of the P initially present in seeds remained in the seed (Table 2). Under P starvation, the smallest difference between P content in seedlings and in initial seeds was found for the low-P tolerant NST. This suggests that this genotype mobilized most of the seed P during the early growth stages. Under P-decient conditions, the highest P concentration in roots was observed for NST, suggesting a priority in the partitioning of P to root development in this genotype. Organic acids exudation and synthesis Organic acids in root exudates Axenic conditions in the root compartment of the culture vessel were maintained until the end of the experiments (18 d). Organic compounds found in the solutions of the root compartment could, therefore, be assumed to be the original exudates and not metabolic products of micro-organisms. Exudates contained monocarboxylic organic acids (acetic, formic, glycolic and lactic acids), dicarboxylic organic acids (malic, oxalic and succinic acids) and tricarboxylic organic acids (citric and trans-aconitic acids). The composition compared well with previous studies of root exudates using maize (Jones and Darrah, 1995; Kraffczyk et al., 1984; Mench et al., 1988; Petersen and Bttger, 1991). Anions of monocarboxylic acids are weak chelators of polyvalent metal cations such as Fe3+ and Ca2+

Results and discussion Plant growth Dry matter production and morphological traits Plant dry matter production of 18 d old seedlings was signicantly higher for the genotype NST than for the other three genotypes (p < 0.001) (Table 1). P deciency signicantly decreased total plant dry matter (p < 0.001) but not root dry matter and increased the root/shoot-ratio (p < 0.001). The effects of P deciency were especially strong for NST: a considerable decrease in plant dry matter (27%) was associated with a strong increase in both root dry matter (56%) and average maximum root length (33%) suggesting increased assimilate allocation in the root system for

257
Table 1. Dry matter production, root/shoot-ratio and maximum root length of 18-d old seedlings. + P = 1 mM P; - P = no P. n = 6. Within the same genotype and the same parameter values followed by the same capital letter are not statistically different at p = 0.05. Within the same P treatment and the same parameter values followed by the same small letter are not statistically different at p = 0.05 Genotype P supply Plant dry matter (g) 0.89 Ab 0.77 Bb 0.98 Ab 0.72 Bb 0.96 Ab 0.78 Bb 1.28 Aa 0.93 Ba Root dry matter (g) 0.29 Aa 0.29 Ab 0.22 Aa 0.26 Ab 0.25 Aa 0.27 Ab 0.25 Ba 0.39 Aa Root / shoot-ratio of dry matter 0.49 Ba 0.60 Ab 0.30 Bb 0.56 Ab 0.35 Bb 0.54 Ab 0.24 Bb 0.73 Aa Average maximum root length (cm) 34.6 Aa 33.4 Ac 33.8 Ba 48.5 Aa 35.6 Aa 39.2 Ab 34.2 Ba 45.4 Aa

Corso

+P P +P P +P P +P P

Sikuani

SA3

NST

and are considered to be inefcient in mobilizing of metal-bound P (Nagarajah, 1970). Monocarboxylic acids were therefore not specically considered in our study. Oxalic acid occurred in very low concentrations and was also ignored. Therefore, the following data specically concentrate on malic, succinic, citric and trans-aconitic acids as well as on the total amount of organic acids. Exudation of organic acids from the whole root system Release of organic acids from the different genotypes was generally higher under P starvation conditions than with 1 mM P treatment (Table 3). Between genotypes, differences in organic acids root exudation existed. The low-P tolerant genotype, NST, showed the highest increase in organic acids root exudation with P deciency. Furthermore, under P-decient conditions, the release of citric and malic acids, both determined in the literature as efcient in the P mobilization from soil (Hofand et al., 1989; Jones and Darrah, 1994b; Nagarajah et al., 1970), was signicantly higher for NST than for the other genotypes (p < 0.001). When expressed in nmol C g1 plant dry weight, trans-aconitic acid was the predominant organic acid in the root exudates of the four maize genotypes (Table 3). The highest rate of exudation of trans-aconitic acid was observed for Corso. While the concentration of trans-aconitic acid in root ex-

udates for NST, SA3 and Sikuani lines was lower than for Corso, the contribution of malic and citric acids in these genotypes was increased. Jones and Darrah (1995) suggested that aconitate may act as a charge-balancing anion in the absence of other organic acids. Succinic acid was only important in root exudates of the acid soil-tolerant line Sikuani. Hue et al. (1986) showed that succinic acid may contribute to Al detoxication by Al-resistant genotypes.

Exudation of organic acids from two different root segments The rate of exudation was different for various root segments (Table 4). It was signicantly higher for root tips (segment A) than for segments B, located at 5 cm distance from the tips (p < 0.001). Release of organic acids from the different root segments was signicantly higher under P starvation conditions than with 1 mM P treatment (p < 0.001) (Table 4). When expressed in nmol C cm1 fresh root, trans-aconitic acid was again the predominant organic acid in the root exudates of the four maize genotypes. For segments A and B, the total exudation of organic acids (p < 0.001), and the release of citric (p = 0.004) and malic acids (p = 0.003) in particular, were signicantly higher for NST than for the other three genotypes. The exudation of succinic acid was only detected from the root tips of Sikuani under P-decient conditions (Table 4).

258
Table 2. P content in 18 d old seedlings grown in P-decient solution (- P) and in 1 mM P solution (+ P). n = 6. Within the same P treatment and the same parameter mean values followed by the same letter are not statistically different at p = 0.05. For each parameter and genotype, mean values from both P treatments were signicantly different at p = 0.05 Genotype P treatment P content (mg/plant) 0.86 b 0.79 b 0.81 b 1.06 a 9.02 b 7.98 b 8.89 b 13.59 a P in roots (mg/g dry weight) 0.99 c 0.99 c 1.14 b 1.35 a 9.57 b 8.94 b 9.99 b 11.49 a P in shoot (mg/g dry weight) 1.19 a 1.14 a 0.99 b 0.99 b 10.47 a 7.89 b 8.99 b 10.41 a Anthocyanin coloration ++ ++++ + +++

Corso Sikuani SA3 NST Corso Sikuani SA3 NST

+P

Table 3. Root exudation of organic acids. + P = 1 mM P; - P = no P. Mean values; n = 6. n.d.: not detected. Means with the same letter are not statistically different at p = 0.05. Capital letters refer to differences between P treatments (read horizontally). Small letters refer to differences between genotypes (read vertically) Genotype Organic acid Root exudation P +P nmol C / g plant dry weight / h 58.1 Ac 4.6 Ab 43.0 Ab 243.8 Aa 395.8 Ab 98.8 Ab 18.8 Aa 36.9 Ac 212.7 Ab 411.4 Ab 86.8 Ab n.d. 47.2 Ab 112.0 Ac 291.0 Ac 133.3 Aa n.d. 69.7 Aa 199.4 Ab 451.2 Aa 37.6 Bc 3.8 Aa 22.9 Bb 252.6 Aa 357.4 Ba 59.6 Bb 2.4 Bb 12.7 Bc 103.1 Bb 215.6 Bb 72.5 Aa 2.2 b 37.8 Aa 67.2 Bc 220.7 Bb 52.3 Bb 0.9 c 22.2 Bb 90.0 Bb 208.9 Bb

Corso

Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids

Sikuani

SA3

NST

259
Table 4. Organic acid exudation from two root segments. Segment A: root tip, length: 1.5 cm; Segment B: distance from root tip: 5 cm, length 1.5 cm. + P = 1 mM P; P = no P. Mean values; n = 6. n.d.: not detected. Means with the same letter are not statistically different at p = 0.05. Capital letters refer to differences between P treatments (read horizontally). Small letters refer to differences between genotypes (read vertically). Roman numbers refer to differences between root segments (read horizontally) Genotype Organic acid Root exudation Segment A Segment B P +P P +P nmol C / cm fresh root / h 0.4 AbI n.d. 0.3 bI 2.5 AbI 3.6 AbI 0.7 AbI 0.3 b 0.3 b 1.9 AbI 3.6 AbI 0.8 AbI n.d. 0.5 AbI 0.8 AbI 2.5 AbI 0.9 AbI n.d. 0.6 AbI 2.2 AbI 4.2 AbI 0.3 Ab n.d. n.d. 2.3 AbI 3.2 AbI 0.6 AbI n.d. n.d. 1.2 BbI 2.1 BbI 0.6 AbI n.d. 0.1 Bb 0.6 AbI 1.4 BbI 0.6 AbI n.d. n.d. 1.3 BbI 2.2 BbI 0.1 bII n.d. 0.1 bI 0.7 AbII 1.1 AbII 0.3 AbII n.d. n.d. 0.6 AbII 1.1 AbII 0.3 AbII n.d. 0.1 bII 0.3 AbII 0.8 AbII 0.4 AbII n.d. 0.1 bII 1.0 AbII 1.7 AbII n.d. n.d. n.d. 0.4 AbII 0.5 BbII 0.2 AbII n.d. n.d. 0.4 AbII 0.7 BbII 0.2 AbII n.d. n.d. 0.2 AbII 0.5 AbII 0.2 AbII n.d. n.d. 0.9 AbI 1.4 AbII

Corso

Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids

Sikuani

SA3

NST

The root release of organic acids did not appear to be associated with the release of protons from maize roots (data not shown), as also showed by Bertrand (1998). This suggests that the mobilization of P in substrates of maize roots may be increased due to the anion exchange and metals complexation of released organic acid anions and not to acidication. Furthermore, as described by Jones and Darrah (1994b), organic acids are dissociated in root cells in the pH conditions of the cytoplasm, and so they should be released as organic anions and should not contribute per se to the acidication of the rhizosphere.

Organic acid content Organic acid content in two different root segments When expressed in mol C per fresh weight, the total content of the organic acids in the root tips (segment A, 01.5 cm) and in the root segments B (56.5 cm) was higher when plant nutrition was P-decient than when it was P-sufcient (p < 0.001) (Table 5). This increase can be related to an increased cation uptake rate, which could not be fully compensated by anion uptake in the absence of phosphate ions, and/or to an increase in NO3 uptake following the onset of P deciency (Imas et al., 1997a, b). Organic acids in tissues are required for counter-balancing increased cation/anion-ratios. Nitrate nutrition leads to an in-

260

Figure 1. Total organic acids content as related to PEPC activity in root tips (1.5 cm). Closed symbols: P-sufcient conditions; open symbols: P-decient conditions. Cor: Corso; Sik: Sikuani. Mean value SE. n = 6.

Figure 2. Total organic acids exudation from roots as related to phloem exudation from shoots. Closed symbols: P-sufcient conditions; open symbols: P-decient conditions. Cor: Corso; Sik: Sikuani. Mean value SE. n = 6.

crease in cytoplasmic pH and the synthesis of organic anions serves as a compensation for the biochemical pH stat and to replace the negative charge lost when NO3 is reduced (Imas et al. 1997a, b; Kirkby and Knight, 1977, Marschner, 1995). Differences in the content of organic acids in segments A and B existed (Table 5). Under P-decient conditions for the four maize lines, organic acids content was equal or lower in root tips than in root segments B. Differences were also noticed between maize genotypes. Total organic acids content, and the content of malic and citric acids in particular were higher in the low-P susceptible SA3 than in the three other genotypes (p < 0.001). Organic acid contents in root tips as related to PEPC-activity Organic acid content in the root tips (segment A) was correlated with PEPC activity (r2 =0.93; Figure 1), suggesting that organic acid content was at least partly controlled by PEPC activity. Root exudation of organic acids from the whole root system and two different root segments as related to organic acid contents Root exudation of organic acids from root segment A (tip, length 1.5 cm) and root segment B (5 cm from the tips, length 1.5 cm) increased with organic acid contents, when expressed both per cm root length and on a fresh weight basis respectively (Tables 4 and 5).

This was especially true for the NST and Sikuani in the whole root system and segment A. However, root exudation by the lines Corso and SA3 was less affected by an increase in organic acid contents. Collectively, the organic acids measurements indicate that under P deciency, a higher content in total organic acids, and malic and citric acids in particular, in segments A and B and a lower root exudation were found for SA3 than for the other lines. It is suggested that the lower rate of exudation of organic acids for SA3 was one reason for the low tolerance of this genotype to P deciency. Organic acids in phloem exudation Trans-aconitic acid was the most abundant organic acid in the phloem exudates of the genotypes Corso, Sikuani and NST (p < 0.001) (Table 6). For SA3, however, malic acid was the most predominant organic acid (p < 0.001). As was suggested above within the root exudation, trans-aconitic may act as a charge-balancing anion in the absence of other organic acids. Under P deciency, the concentration of organic acids signicantly increased in the phloem exuded from the shoot (p < 0.001). NO3 reduction and assimilation in the shoots is very important for maize (Marschner, 1995; Pearson et al., 1981), suggesting that a high proportion of organic acids may be synthesized in the shoots and transported to the roots through phloem, where it contributes to root exudation. For rape, Hofand et al. (1990) showed that the secretion of organic acids by the roots of P-decient resulted

261
Table 5. Organic acid content of two root segments. Segment A: root tip, length: 1.5 cm; Segment B: distance from root tip: 5 cm, length 1.5 cm. + P = 1 mM P; P = no P. Mean values; n = 6. n.d.: not detected. Means with the same letter are not statistically different at p = 0.05. Capital letters refer to differences between P treatments (read horizontally). Small letters refer to differences between genotypes (read vertically). Roman numbers refer to differences between root segments (read horizontally) Genotype Organic acid Root content Segment A Segment B P +P P +P mol C / g root fresh weight 12.4 AdI 1.3 AbI 11.5 BaI 133.9 AaI 184.9 AbI 39.8 AcI 7.6 AaI 39.7 AaI 71.1 AcII 183.0 AbI 68.1 AaI 0.8 AbI 38.4 BaII 87.8 AbII 225.8 AaII 55.6 AbI 0.8 AbI 14.4 AaI 93.2 AbII 190.6 AbII 7.2 BcI 0.8 AbI 21.7 AbI 99.3 BaI 146.7 BaI 29.1 BbI 4.8 BaI 16.1 BbI 49.0 BbII 116.0 BbI 58.2 AaI 0.9 AbI 47.0 AaI 33.2 BcII 120.6 BbII 36.0 BbI 0.4 AbI 3.6 BcII 46.0 BbI 109.6 BbI 13.2 AcI 0.3 AbII 7.8 BbI 143.7 AbI 191.0 AcI 35.6 AbI 6.8 AaI 8.4 AbII 107.3 AcI 184.8 AcI 71.3 AaI 0.3 AbII 112.0 AaI 187.0 AaI 315.7 AaI 39.8 AbII 0.4 AbI 7.7 BbII 167.1 AaI 245.8 AbI 4.0 BcII 0.4 AbI 13.8 AcII 89.8 BaI 120.9 BbI 24.0 BbI 4.4 BaI 6.0 BdII 68.6 BaI 117.1 BbI 48.7 BaI 0.5 AbI 24.0 BbII 80.9 BaI 169.9 BaI 19.3 BbII 0.4 AbI 31.3 AaI 29.5 BbII 78.0 BcII

Corso

Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids

Sikuani

SA3

NST

from an increase in the PEP carboxylase activity in the shoot under P-decient conditions, which also led to the accumulation of citrate in the shoot and a higher citrate/sugar ratio in the phloem. Root exudation as related to phloem exudation of shoots The organic acids content in the phloem exudates of shoots was related to root exudation (Figure 2). Under P deciency, organic acids increased both in roots and phloem exudates, suggesting that the organic acids synthesized in shoots may be transferred to the roots and exuded. Nevertheless, as also observed for the organic acids content in roots (Table 5), the total release of organic acids from phloem, malic and citric acids especially,

was signicantly higher for SA3 than for the other lines (p < 0.001) (Table 6). These results suggest that for maize the transfer of organic acids from the shoot (via the phloem) to the roots does not control the tolerance of some genotypes to low-P conditions. Acid phosphatase Acid phosphatase activity was measured on the root surface and was signicantly lower in Corso than in the three other maize lines (p < 0.001) (Table 7). Activity increased strongly under P deciency in the low-P tolerant NST line (p = 0.002). Only small effects of P deciency were found for SA3 (p = 0.36) and Sikuani (p = 0.13). The results suggest that the high acid phosphatase activity in NST under P de-

262
Table 6. Organic acids released in phloem from shoot. + P = 1 mM P; P = no P. Mean values; n = 6. n.d.: not detected. Means with the same letter are not statistically different at p = 0.05. Capital letters refer to differences between P treatments (read horizontally). Small letters refer to differences between genotypes (read vertically) Genotype Organic acid Phloem shoot exudation P +P nmol C / g plant dry weight / h 174.5 Ac 14.3 Ab 108.1 Ad 1005.7 Aa 1444.6 Ab 389.5 Ab 73.7 Aa 153.4 Ac 887.4 Ab 1674.9 Aa 667.1 Aa n.d. 289.4 Aa 566.6 Ac 1716.6 Aa 399.9 Ab n.d. 216.2 Ab 591.9 Ac 1356.7 Ab 188.9 Ac 12.5 Aa 92.6 Ac 1027.1 Aa 1380.1 Ab 416.5 Ab 15.9 Ba 88.2 Bc 711.7 Bb 1338.3 Bb 575.4 Ba 17.6 a 275.5 Aa 488.5 Bd 1509.2 Ba 388.1 Ab 6.2 b 165.1 Bb 608.8 Ac 1232.7 Ac

Corso

Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids Malic Succinic Citric Trans-aconitic Total org. acids

Sikuani

SA3

NST

Table 7. Acid phosphatase activity as released into solution and as adhering to root surface of 18 days old seedlings. Mean value; n = 6. + P = 1 mM P; P = no P; mU = nmol P / min. Means with the same letter are not statistically different at p = 0.05. Capital letters refer to differences between P treatments (read horizontally). Small letters refer to differences between genotypes (read vertically). Genotype Acid phosphatase activity Released into solution Adhering to root surface P +P P +P mU / g root dry weight / day mU / g root dry weight 11.9 Ac 15.8 Ab 9.2 Ad 19.5 Aa 11.4 Ab 13.7 Ba 7.7 Bc 10.6 Bb 203 Ad 319 Ab 281 Ac 398 Aa 212 Ab 294 Aa 274 Aa 306 Ba

Corso Sikuani SA3 NST

263 ciency may contribute to the low-P tolerance of this genotype. Released acid phosphatase activity signicantly increased under P deciency (p < 0.001) and was maximum for the low-P tolerant NST line. The acid phosphatase activity in the NaCl eluates was higher than in the nutrient solution and may have originated from acid phosphatase adhering to the root epidermal cell layers, as suggested by Tadano and Sakai (1991). We propose that the higher tolerance of the genotype NST to low-P conditions may be related to 1. a high utilization of the seed P by the young seedling plant, 2. a relatively high root dry matter and greater root length, to explore a larger volume of soil, 3. a high anthocyanin coloration, as a protection against oxygen free radical stress, 4. exudation of large amounts of citric and malic acids, both known to be efcient for soil P mobilisation, and 5. a high acid phosphatase activity which may promote P acquisition from organically bound P.

Conclusions P deciency in hydroponic culture resulted in decreased dry matter production of the four maize genotypes. The decrease was especially evident in the low-P tolerant NST and the acid-tolerant Sikuani, and was accompanied by increases in root/shoot-ratio and in average maximum root length. This response appeared to be related to an increased investment of plant resources in root growth to improve P acquisition. Organic acids contents in root tips were increased under P deciency and were closely related to PEPC activity. Differences in organic acids contents between genotypes were not related to their low-P tolerance. Root exudation of organic acids increased with root contents as P supply was varied for the genotypes NST and Sikuani. On the other hand, root exudation was not related to root content in the genotypes Corso and SA3. The low exudation of organic acids by SA3, despite high root contents, may contribute to the susceptibility of this maize genotype to low-P conditions. Under P deciency, organic acids content increased in phloem sap released from shoots, cocommittant with root exudates. Shootroot transport of organic acids may contribute to root exudation. Nevertheless, as this transfer of organic acids, malic and citric acids in particular, was higher for SA3 than for NST, the contribution of organic acids synthesized in the shoot might only partly explain the response of low-P tolerant maize genotypes to P deciency. There was a difference between genotypes in the organic acids composition of roots and root exudates. Trans-aconitic acid and malic acid were predominant in phloem exudates from the shoot, in roots and in root exudates of the four maize genotypes. Root acid phosphatase activity was higher in the lines NST and Sikuani than in the genotypes Corso and SA3 and may be important for low-P tolerance of these genotypes. Acknowledgements The authors thank Dr D. K. Friesen from the CIMMYT regional ofce at ICRAF, Nairobi, Kenya and Dr S. Pandey from the CIMMYT regional ofce at CIAT, Cali, Colombia, for generously providing the seeds of the maize genotypes ICA V-110 Sikuani.

References
Anghinoni I and Barber S A 1980 Phosphorus inux and growth characteristics of corn roots as inuenced by phosphorus supply. Agron. J. 72, 685688. Bertrand I 1998 Importance of the protons release on the mobilisation of mineral phosphorus and iron by roots. Study of models minerals: calcite and goethite. Ph.D Thesis, Universit Aix-Marseille III, France. Braum S M and Helmke P A 1995 White lupin utilizes soil phosphorus that is unavailable to soybean. Plant Soil 176, 95100. Friesen D K, Rao I M, Thomas R J, Oberson A and Sanz J I 1997 Phosphorus acquisition and cycling in crop and pasture systems in low fertility tropical soils. Plant Soil 196, 289294. Gardner W K, Barber D A and Parbery D G 1983 The acquisition of phosphorus by Lupinus albus L. III. The probable mechanism by which phosphorus movement in the soil/root interface is enhanced. Plant Soil 70, 107124. Gerke J 1992 Phosphate, aluminum and iron in the soil solution of three different soils in relation to varying concentrations of citric acid. Z. Panzenernaehr. Bodenk. 155, 339343. Helal H M and Sauerbeck D 1987 Phosphatase-Aktivitt von Panzenwurzeln und Bden in Abhngigkeit von der P-Versorgung. VDLUFA-Schriftenreihe 23, 195201. Hofand E, Findenegg G R and Nelemans J A 1989 Solubilization of rock phosphate by rape. Plant Soil 113, 155160. Hofand E, Nelemans J A and Findenegg G R 1990 Origin of organic acids exuded by roots of phosphorus stressed rape (Brassica napus) plants. In Plant Nutrition-Physiology and Applications. Ed. ML Van Beusichem, pp 179183. Kluwer Academic Publishers, Norwell. Hoagland, D R and Arnon I R 1938 The water culture method for growing plants without soils. Circ. Calif. Agric. Exp. Stn. No. 347. Hrazdina G and Zobel A M 1991 Cytochemical localization of enzymes in plant cells. Bot. Rev. 129, 269322.

264
Hue N V, Craddock G R and Adams F 1986 Effect of organic acids on Al toxicity in subsoils. Soil Sci. Soc. Am. J. 50, 2834. Imas P, Bar-Yosef B, Kafka U and Ganmore-Neumann R 1997a Release of carboxylic anions and protons by tomato roots in response to ammonium nitrate ratio and pH in nutrient solution. Plant Soil 191, 2734. Imas P, Bar-Yosef B, Kafka U and Ganmore-Neumann R 1997b Phosphate induced carboxylate and proton release by tomato roots. Plant Soil 191, 3539. John M K 1970 Colorimetric determination of phosphorus in soil and plant materials with ascorbic acid. Soil Sci. 4, 214220. Johnson J F, Vance C P and Allan D L 1996 Phosphorus deciency in Lupinus albus. Altered lateral root development and enhanced expression of phosphoenolpyruvate carboxylase. Plant Physiol. 112, 3141. Jones D L and Darrah P R 1994a Amino-acid inux at the soil-root interface of Zea mays L. and its implications in the rhizosphere. Plant Soil 163, 112. Jones D L and Darrah P R 1994b Role of root derived organic-acids in the mobilization of nutrients from the rhizosphere. Plant Soil 166, 247257. Jones D L and Darrah P R 1995 Inux and efux of organic acids across the soilroot interface of Zea mays L. and its implications in rhizosphere C ow. Plant Soil 173, 103109. Khamis S, Chaillou S and Lamaze T 1990 CO2 assimilation and partitioning of carbon in maize deprived of orthophosphate. J. Exp. Bot. 41, 16191625. King R W and Zeevaart J A D 1974 Enhancement of phloem exudation from cut petioles by chelating agents. Plant Physiol. 53, 96103. Kirkby E A and Knight A H 1977 Inuence of the level of nitrate nutrition on ion uptake and assimilation, organic acid accumulation, and cation-anion balance in whole tomato plants. Plant Physiol. 60, 349353. Kraffczyk I, Trolldenier G and Beringer H 1984 Soluble roots exudates of maize: Inuence of potassium supply and rhizosphere micro-organisms. Soil Biol. Biochem. 16, 315322. Kummerova M 1986 Localization of acid phosphatase in maize root under phosphorus deciency. Biol. Plant. 28, 270274. Lynch J P 1995 Root architecture and plant productivity. Plant physiol. 109, 713. Lynch J M and Whipps J M 1989 Substrate ow in the rhizosphere. In The Rhizosphere and Plant Growth. Eds. DL Keister and PB Cregan, pp 1524. Kluwer Academic Publishers, Dortrecht, The Netherlands. Marschner H 1995 Mineral nutrition of higher plants. Academic Press, London 2nd edn. 889 p. Matsumoto H, Okada K and Takahashi E 1979 Excretion products of maize roots from seedling to seed development stage. Plant Soil 53, 1726. McCully M E 1999 Roots in soil: Unearthing the complexities of roots and their rhizospheres. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 695718. Mench M, Morel J L, Guckert A and Guillet B 1988 Metal binding with root exudates of low molecular weight. J. Soil Sci. 39, 521 527. Mollier A and Pellerin S 1999 Maize root system growth and development as inuenced by phosphorus deciency. J. Exp. Bot. 55 (333), 487497. Nagarajah S, Posner A M and Quirk J P 1970 Competitive adsorptions of phosphate with polygalacturonate and other organic anions on kaolinite and oxide surfaces. Nature (London) 228, 8384. Pearson C J, Volk R J and Jackson W A 1981 Daily changes in nitrate inux, efux and metabolism in maize and pearl millet. Planta 152, 319324. Petersen W and Bttger M 1991 Contribution of organic acids to the acidication of the rhizosphere of maize seedlings. Plant Soil 132, 159163. Raghothama K G 1999 Phosphate acquisition. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 665693. Rosolem C A, Assis J S and Santiago A D 1994 Root growth and mineral nutrition of corn hybrids as effected by phosphorus and lime. Com. Soil Sci. Plant Anal. 25, 24912499. Schopfer P 1984 Photomorphogenesis. In Advanced Plant Physiology. Ed. MB Wilkins, pp 380407. Pitman, London, Marsheld, Melbourne. Staunton S and Leprince F 1996 Effect of pH and some organic anions on the solubility of soil phosphate: implications for P bioavailability. Eur. J. Soil. Sci. 47, 231239. SYSTAT 1994 SPSS Inc., Chicago. Tabatabai M A 1982 Soil enzymes. In Methods of Soil Analysis. Part 2, Chemical and Microbiological Properties. Eds. AL Page, RH Miller and DR Keeney, pp 923931. Agronomy ASA, Madison, Wisconsin, USA. Tadano T and Sakai H 1991 Secretion of acid phosphatase by the roots of several crop species under phosphorus-decient conditions. Soil Sci. Plant Nutr. 37 (1), 129140. Tarafdar J C and Jungk A 1987 Phosphatase activity in the rhizosphere and its relation to the depletion of soil organic phosphorus. Biol. Fertil. Soils 3, 199204. Uren N C and Reisenauer H M 1988 The role of root exudates in nutrient acquisition. Adv. Plant Nutr. 3, 79114. Vance C P, Stade S and Maxwell C A 1983 Alfalfa root nodule carbon dioxide xation. I. Association with nitrogen xation and incorporation into amino acids. Plant Physiol. 72, 469473. Zhang F S, Ma J and Cao Y P 1997 Phosphorus deciency enhances root exudation of low-molecular weight organic acids and utilization of sparingly soluble inorganic phosphates by radish (Raghanus satiuvs L.) and rape (Brassica napus L.) plants. Plant Soil 196, 261-264.